title: Bottom-up proteome analysis of E. coli using capillary zone electrophoresis-tandem mass spectrometry with an electrokinetic sheath-flow electrospray interface.
abstract: The Escherichia coli proteome was digested with trypsin and fractionated using SPE on a C18 SPE column. Seven fractions were collected and analyzed by CZE-ESI-MS/MS. The separation was performed in a 60-cm-long linear polyacrylamide-coated capillary with a 0.1% v/v formic acid separation buffer. An electrokinetic sheath-flow electrospray interface was used to couple the separation capillary with an Orbitrap-Velos operating in higher-energy collisional dissociation mode. Each CZE-ESI-MS/MS run lasted 50 min and total MS time was 350 min. A total of 23 706 peptide spectra matches, 4902 peptide IDs, and 871 protein group IDs were generated using MASCOT with false discovery rate less than 1% on the peptide level. The total mass spectrometer analysis time was less than 6 h, the sample identification rate (145 proteins/h) was more than two times higher than previous studies of the E. coli proteome, and the amount of sample consumed (<1 μg) was roughly fourfold less than previous studies. These results demonstrate that CZE is a useful tool for the bottom-up analysis of prokaryote proteomes.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, USA.
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Yan
authors.forename: David C
authors.initials: DC
authors.lastname: Essaka
authors.forename: Liangliang
authors.initials: L
authors.lastname: Sun
authors.forename: Guijie
authors.initials: G
authors.lastname: Zhu
authors.forename: Norman J
authors.initials: NJ
authors.lastname: Dovichi
mesh_terms: D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism*; D020543:Proteome / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
chemical_list: D020543:Proteome
keywords: Bottom-up proteomics; CZE-ESI-MS/MS; Electrokinetically driven sheath flow CE-MS interface; Escherichia coli; Technology